Bacteria play important roles in human health. Complex bacterial communities are important components of normal physiology, as is increasingly being understood through investigations of human microbiota. Other bacteria are well known as pathogens that can cause life-threatening infections. Whether impacting health or disease, interactions of microbes with hosts share many fundamental features. Paramount among these is the ability of bacteria to monitor their intracellular and extracellular environment and to elicit appropriate adaptive responses to changing conditions. The most prevalent mechanism for coupling stimuli to output responses is a two-component system phosphotransfer pathway involving a sensor histidine protein kinase and a phosphorylation-activated response regulator that generates the output response. This versatile regulatory scheme has been exploited in >200,000 systems identified to date. While core features, specifically, the structures and biochemical activities of individual domains of histidine kinases and response regulators, are conserved, there is great variation in how they are configured to optimize specific needs of individual systems. Two decades of research have focused on biochemical characterization of two-component proteins in vitro, revealing autophosphorylation, phosphatase, phosphotransfer, and autodephosphorylation activities that differ greatly in magnitude among homologous proteins in different systems. An understanding of how these parameters relate to function within cells has been hindered by an inability to directly measure labile protein phosphorylation in vivo. This project will utilize recently developed methodologies that enable such measurements to establish fundamental principles of how protein levels, enzyme activities, and pathway architecture are configured to optimize both fitness and behavior. The existing methodologies will be extended to kinetic characterization of response regulator phosphorylation allowing, for the first time, determination of the rates of the opposing kinase and phosphatase activities of histidine kinases within cells, and how these rates are altered by stimuli. Previous studies have shown that system output is highly dependent on the phosphatase activity of histidine kinases. However, characterization of this activity and its enzymatic mechanism lags behind all other conserved activities of two-component proteins. This project will address this issue by using combined structural and biochemical approaches to define the enzymatic mechanisms of phosphatase activity in the two major classes of histidine kinases, HisKA and HisKA_3. Together, the proposed studies will fill critical gaps in current knowledge, enable new approaches for investigating two-component signaling system configuration, determine how previously established in vitro parameters relate to system behavior in vivo, and define fundamental principles with predictive capabilities for understanding many as yet unexplored two-component systems. This fundamental knowledge of microbial regulation that is essential to host-microbe interactions will provide a firm underpinning for development of both probiotic and antibiotic therapeutic strategies.